In 2013, antibiotic resistant bacteria were responsible for an estimated 2 million illnesses and at least 23,000 deaths in the United States alone {(CDC), 2013 #1948}. Diversification of our antibiotic arsenal is crucial for counteracting the spread of resistance and ensuring our ability to effectively treat bacterial infections. The vast majority of antibiotics, whose structural complexity presents a considerable challenge to chemical synthesis, are produced industrially by engineered microbial hosts {Elander, 2003 #1943; Hamed, 2013 #1882}. Unfortunately, many species cannot be successfully engineered to produce antibiotics at amounts sufficient for an industrial process. For many compounds, biosynthesis pathways can be transferred to genetically-tractable, well-established industrial production species such as Escherichia coli {Keasling, 2010 #166}, which can be further modified with enzymes from different biosynthetic pathways, potentially generating enormous diversity {Weeks, 2011 #1840}. Unfortunately, this solution is not readily available to antibiotic manufacture due to the obvious toxicity of the compounds: the host cells would be destroyed by their products.
Persister cells are bacteria in a transient, growth-arrested state that provides tolerance of multiple classes of antibiotics (1, 2). The ability to resist antibiotic treatment enables persister cells to sustain bacterial infections. The persistent state, which likely describes a number of different growth-arrested physiologies (3), has been at least partially attributed to the activity of toxin/antitoxin (TA) modules (4). TA modules are composed of two genes; one of the two genes encodes a toxin protein whose expression slows or stops cell growth, and the other encodes its corresponding antitoxin protein or RNA, which either inactivates its toxin directly or prevents translation of the toxin protein. Toxins have diverse enzymatic activities and cellular targets (5), and it is thought that active toxins trigger the growth slowdown or arrest that is characteristic of the persistent state by inhibiting a central cellular process. A link between TA modules and persistence has recently been demonstrated in a laboratory setting using Escherichia coli MG1655. In that study, deletion of five or more chromosomally encoded TA modules reduced the number of persister cells generated (6).
The first toxin protein to be linked to persistence in E. coli was the serine/threonine kinase HipA (7), which forms a TA module with its cognate antitoxin, HipB. HipA overexpression within growing E. coli bacteria causes multiple responses that are the hallmarks of persistence, including growth arrest and tolerance of certain classes of antibiotics, as well as attenuation of DNA replication, transcription, and translation (8). Some insight into the mechanism of HipA-induced growth arrest was gained when it was discovered that HipA phosphorylates the protein EF-Tu (9), an essential translation factor that catalyzes the binding of aminoacyl-tRNA to the ribosome. Phosphorylation by HipA is expected to deactivate EF-Tu (10), which may account for the inhibition of translation within HipA-arrested cells. How HipA expression also inhibits DNA and RNA synthesis and provides tolerance of certain antibiotics when translation is inhibited is unclear. Translational inhibition is insufficient to explain the general arrest of macromolecular synthesis, as translation inhibition by other means (e.g., by addition of the ribosome inhibitor chloramphenicol) does not inhibit DNA replication or RNA transcription (11) or provide antibiotic tolerance (12). While it is possible that HipA inhibits DNA and rRNA synthesis directly, such as by phosphorylating protein targets other than EF-Tu, we and others have observed that the growth arrest triggered by HipA is similar to the effects of the alarmone guanosine tetraphosphate (ppGpp) (8). ppGpp allosterically inhibits enzymes that are central to an incredible variety of cellular processes in E. coli, including priming of DNA replication (13), rRNA transcription (14), translation (15), phospholipid synthesis (16), and certain metabolic enzymes (17). A link between the stringent response and persistence has been postulated on the basis of the finding that an E. coli strain lacking the enzymes required to synthesize ppGpp (RelA and SpoT) generates fewer persister cells (18). One possible interpretation of this finding might be that ppGpp is necessary for inducing a cellular response, such as expression of toxins or degradation of antitoxins, that triggers persistence upon application of stress. An alternative explanation is that ppGpp directly confers resistance by inhibiting cellular processes itself (18), an aspect of regulation by ppGpp that is often unappreciated (19). Understanding the mechanisms by which toxin activity leads to growth arrest and antibiotic tolerance is critical for informing efforts to eradicate persisters (3).